Prolonged cardiac overexpression of the mitochondrial biogenesis regulatory transcriptional coactivator PGC-1α disrupts cardiac contractile function and its genetic ablation limits cardiac capacity to enhance workload. In contrast, transient induction of PGC-1α alleviates neuronal cell oxidative stress and enhances skeletal myotube anti-oxidant defenses. We explored whether transient upregulation of PGC-1α in the heart protects against ischemia–reperfusion injury. The transient induction of PGC-1α in the cardiac-restricted inducible PGC-1α transgenic mouse, increased PGC-1α protein levels 5-fold. Following 25min of ischemia and 2h of reperfusion on a Langendorff perfusion apparatus, contractile recovery and the rate pressure product was significantly blunted in mice overexpressing PGC-1α vs. controls. Affymetrix gene array analysis showed a 3-fold PGC-1α-mediated upregulation of adenine nucleotide translocase 1 (ANT1). As ANT1 upregulation induces cardiomyocyte cell death we investigated whether the induction of ANT1 by PGC-1α contributes to this enhanced ischemia-stress susceptibility. Infection with adenovirus harboring PGC-1α into cardiac-derived H9c2 cells significantly upregulates ANT1 without changing basal cell viability. In response to anoxia-reoxygenation injury cell death is significantly increased following PGC-1α overexpression. This detrimental effect is abolished following siRNA knockdown of ANT1. Similarly, the attenuation of ANT-1 in the presence of PGC-1α overexpression preserves the mitochondrial membrane potential in response to hydrogen-peroxide stress. Interestingly, the isolated knockdown of ANT1 also protects H9c2 cells from anoxia-reoxygenation injury. Taken together these data suggest that transient induction of PGC-1α in the murine heart decreases ischemia–reperfusion contractile recovery and diminishes anoxia-reoxygenation tolerance in H9c2 cells. These adverse phenotypes appear to be mediated, in part, by PGC-1α induced upregulation of ANT1.